Browsing by Author "Shemanko, Carrie S."
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- ItemOpen AccessCharacterization of Rapid Nongenomic Cortisol Signalling in Rainbow Trout Liver(2018-08-03) Thraya, Marwa; Vijayan, Mathilakath M.; Habibi, Hamid R.; Shemanko, Carrie S.Glucocorticoids are critical in the regulation of metabolic processes for stress adaptation. Cortisol, the primary glucocorticoid in fish, exerts physiological effects through genomic and nongenomic signalling pathways. The genomic response to cortisol is characterized by activation of the corticosteroid receptors, glucocorticoid receptor (GR) and mineralocorticoid receptor (MR), whereas the nongenomic cortisol response involves rapid changes to downstream second-messenger signalling cascades independent of gene regulation. However, nongenomic glucocorticoid signalling remains poorly characterized in teleost models and even more so in the liver, despite the importance of this tissue in regulating both metabolic and physiological adjustments in the face of a stressor. Therefore, the primary objective of this thesis was to further elucidate mechanisms of rapid cortisol signalling in rainbow trout (Oncorhynchus mykiss) liver. The hypothesis tested was that activation of secondary-signalling cascades would mediate rapid cortisol effects and glucocorticoid-membrane receptor(s) were involved in initiating this response. Indeed, stress and the attendant rise in cortisol rapidly modulated cAMP response element binding protein (CREB) phosphorylation, and reduced reactive oxygen species (ROS) generation and glutathione production in vivo. While corticosteroid receptors were identified on the plasma membrane, RU486 had no effect on CREB signalling or ROS generation in vitro, suggesting that these endpoints are GR-independent. Also, cortisol did not rapidly affect the transcript abundance of glucocorticoid-responsive genes associated with the corticosteroid receptors along with biomarkers for immune, growth, metabolic, and oxidative stress systems, suggesting a role for genomic activation in directly regulating the stress response. Furthermore, a membrane-specific cortisol receptor(s) remains unidentified in any species. Altogether, the results presented here propose a role for rapid nongenomic glucocorticoid signalling in modulating the stress-mediated metabolic responses in fish liver, and this provides a novel approach in studying the role of glucocorticoids in mediating stress coping mechanisms.
- ItemOpen AccessComparative proteomic analysis of proliferating and functionally differentiated mammary epithelial cells(American Society for Biochemistry and Molecular Biology Inc., 2003) Desrivieres, S.; Prinz, T.; Palomino-Laria, N. C.; Meyer, M.; Boehm, G.; Schafer, J.; Neumann, T.; Groner, B.; Shemanko, Carrie S.
- ItemOpen AccessCytotoxicity, cellular localization and photophysical properties of Re(I) tricarbonyl complexes bound to cysteine and its derivatives(Springer Nature, 2020-06-24) Capper, Miles S.; Enriquez Garcia, Alejandra; Macia, Nicolas; Lai, Barry; Lin, Jian-Bin; Nomura, Masaharu; Alihosseinzadeh, Amir; Ponnurangam, Sathish; Heyne, Belinda; Shemanko, Carrie S.; Jalilehvand, FaridehThe potential chemotherapeutic properties coupled to photochemical transitions make the family of fac-[Re(CO)3(N,N)X]0/+ (N,N = a bidentate diimine such as 2,2'-bipyridine (bpy); X = halide, H2O, pyridine derivatives, PR3, etc.) complexes of special interest. We have investigated reactions of the aqua complex fac-[Re(CO)3(bpy)(H2O)](CF3SO3) (1) with potential anticancer activity with the amino acid l-cysteine (H2Cys), and its derivative N-acetyl-l-cysteine (H2NAC), as well as the tripeptide glutathione (H3A), under physiological conditions (pH 7.4, 37 °C), to model the interaction of 1 with thiol-containing proteins and enzymes, and the impact of such coordination on its photophysical properties and cytotoxicity. We report the syntheses and characterization of fac-[Re(CO)3(bpy)(HCys)]·0.5H2O (2), Na(fac-[Re(CO)3(bpy)(NAC)]) (3), and Na(fac-[Re(CO)3(bpy)(HA)])·H2O (4) using extended X-ray absorption spectroscopy, IR and NMR spectroscopy, electrospray ionization spectrometry, as well as the crystal structure of {fac-[Re(CO)3(bpy)(HCys)]}4·9H2O (2 + 1.75 H2O). The emission spectrum of 1 displays a variance in Stokes shift upon coordination of l-cysteine and N-acetyl-l-cysteine. Laser excitation at λ = 355 nm of methanol solutions of 1–3 was followed by measuring their ability to produce singlet oxygen (1O2) using direct detection methods. The cytotoxicity of 1 and its cysteine-bound complex 2 was assessed using the MDA-MB-231 breast cancer cell line, showing that the replacement of the aqua ligand on 1 with l-cysteine significantly reduced the cytotoxicity of the Re(I) tricarbonyl complex. Probing the cellular localization of 1 and 2 using X-ray fluorescence microscopy revealed an accumulation of 1 in the nuclear and/or perinuclear region, whereas the accumulation of 2 was considerably reduced, potentially explaining its reduced cytotoxicity.
- ItemOpen AccessDNA based prenatal testing for the skin blistering disorder epidermolysis bullosa simplex(John Wiley & Sons, 2000) Rugg, E. L.; Baty, D.; Shemanko, Carrie S.; Magee, G.; Polak, S.; Bergman, R.; Kadar, T.; Boxer, M.; Falik-Zaccai, T.; Borochowitz, Z.; Lane, E. B.
- ItemOpen AccessEpidermolysis bullosa simplex in Scotland is caused by a spectrum of keratin mutations(Nature Publishing Group, 2007) Rugg, E. L.; Horn, H. M.; Smith, F. J.; Wilson, N. J.; Magee, G.; Shemanko, Carrie S.; Tidman, M. J.; Lane, E. B.
- ItemOpen AccessGeneration and characterization of cell lines expressing EBS keratin mutations: scratch assays show faster migration with more disruptive mutations(Blackwell Publishing, 2003) Morley, S. M.; D'Alessandro, M.; Sexton, C.; Rugg, E. L.; Navsaria, H.; Shemanko, Carrie S.; Hohl, D.; Heagerty, A. I.; Leigh, I. M.; Lane, E. B.
- ItemOpen AccessHeat shock protein-90-alpha, a prolactin-STAT5 target gene identified in breast cancer cells, is involved in apoptosis regulation(BioMed Central, 2008-11-13) Perotti, Christian; Liu, Ruixuan; Parusel, Christine T.; Böcher, Nadine; Schultz, Jörg; Bork, Peer; Pfitzner, Edith; Groner, Bernd; Shemanko, Carrie S.
- ItemOpen AccessLaryngeal involvement in the Dowling-Meara variant of epidermolysis bullosa simplex with keratin mutations of severely disruptive potential(Blackwell Publishing, 2000) Shemanko, Carrie S.; Horn, H. M.; Keohane, S. G.; Hepburn, N.; Kerr, A. I.; Atherton, D. J.; Tidman, M. J.; Lane, E. B.
- ItemOpen AccessMultifactorial Control of Reproductive and Growth Axis in Goldfish: Influence of Neuropeptides and Thyroid Hormone(2019-09-16) Ma, Yifei; Habibi, Hamid R.; Chang, John P.; Shemanko, Carrie S.Reproduction and growth are under multifactorial control of neurohormones and peripheral hormones. The main regulator of reproduction in vertebrates is gonadotropin-releasing hormone (GnRH) which has been shown to stimulate both gonadotrope and somatotrope activity in the pituitary. Other neuropeptides like gonadotropin-inhibitory hormone (GnIH) also play important roles in regulation of growth and reproduction. Although, information regarding the sex related effects on GnRH and GnIH is rather limited as the majority of studies are based on mixed sex fish. Thyroid hormones are important regulators of metabolism but have shown to exert seasonal influence on reproduction as well. Changes in steroid and peptide hormone levels are often associated with seasonal shifts between growth and reproduction. These changes occur at the level of hypothalamus and pituitary, as well as peripheral tissues. Previous studies have demonstrated that GnRH, GnIH, and T3 are important factors contributing to central and peripheral regulation of multifactorial reciprocal control of growth and reproduction in a number of species. The purpose of this thesis was to study seasonally related effects of GnRH, GnIH, and T3 on reproductive and growth axis in male and female goldfish at three stages of gonadal recrudescence: sexually regressed stage, mid recrudescence, and late recrudescence. The effects of GnRH, GnIH, and T3 injection treatments were evaluated by measuring circulating LH and GH concentrations, as well as transcript levels for several genes involved in the regulation of reproduction and growth in goldfish. In females and males, GnRH is a crucial stimulator of both GH and LH secretion with seasonally related effects. In males and females, GnIH stimulated basal GH concentrations in gonadally regressed fish, and GnIH inhibitory action on GnRH-induced LH response was observed in late, but not in mid recrudescence. T3 actions on basal and GnRH- or GnIH-induced GH secretion were generally inhibitory, depending on season in female goldfish. Whereas T3 treatment mainly had stimulatory effects on circulating LH levels and inhibitory effects on serum GH concentrations in males. In the liver and gonads, we observed seasonal differences in thyroid receptors, estrogen receptors, vitellogenin, follicle-stimulating hormone receptor, aromatase and IGF-I transcript levels that were tissue- and sex-specific. These seasonal expression patterns clearly follow seasonal reproductive activity in both males and females. Generally, there were no clear correlation between circulating LH and GH levels and peripheral transcript levels, presumably due to possible direct interaction of GnRH and GnIH at the level of liver, testes or ovaries in addition to indirect effects of GH, LH and FSH levels. The findings in this thesis support the hypothesis that GnRH and GnIH are important components of multifactorial mechanisms that work in concert with T3 to regulate reciprocal control of reproduction and growth in goldfish. Furthermore, these results provide important information on the sex specific functions of GnRH, GnIH, and T3 during the seasonal cycle at the level of pituitary secretion as well as liver and gonads. The overall results provide insight into mechanisms controlling reciprocal control of growth and reproductive function in male and female goldfish.
- ItemOpen AccessMutations in the plakophilin 1 gene result in ectodermal dysplasia/skin fragility syndrome(Nature Publishing Group, 1997) McGrath, J. A.; McMillan, J. R.; Shemanko, Carrie S.; Runswick, S. K.; Leigh, I. M.; Lane, E. B.; Garrod, D. R.; Eady, R. A.
- ItemOpen AccessA Novel Association Between Glutamine Metabolism and Clinical Progression in DCMA, A Mitochondrial Cardiomyopathy(2020-02-27) King, Melissa Anne; Lewis, Ian A.; Greenway, Steven C.; Shemanko, Carrie S.; Zaremberg, VaninaThe Dilated Cardiomyopathy with Ataxia Syndrome (DCMA) is a severe mitochondrial disorder with a high rate of mortality. DCMA is caused by mutations in the poorly characterized DNAJC19 gene which cause a variety of clinical characteristics including elevated 3-methylglutaconic and 3-methylglutaric acids. How these mutations elicit the complex disease presentation is unclear. For this thesis, I developed a fibroblast-based model to study DCMA mitochondrial metabolism. Using stable isotope labeling and metabolomics, I demonstrate that DCMA may be linked to alterations in glutamine catabolism and propose a new model for DCMA involving an inability to import glutamate into the mitochondria. Furthermore, I show that the metabolic phenotype correlates with patient clinical progression. This novel finding could allow clinicians to predict DCMA patient outcomes. This thesis paves the way to reclassifying DCMA as a disorder of glutamine metabolism, and may guide more effective diagnostics for preventing the early death of affected children.
- ItemOpen AccessNuclear localization of dirhodium(II) complexes in breast cancer cells by X-ray fluorescence microscopy(The Royal Society of Chemistry, 2019-06-05) Enriquez Garcia, Alejandra; Lai, Barry; Gopinathan, Sesha Gopal; Harris, Hugh H.; Shemanko, Carrie S.; Jalilehvand, FaridehThe cellular distribution of three dirhodium(II) complexes with a paddlewheel structure was investigated using synchrotron-based X-ray fluorescence microscopy and cell viability studies. Complexes with vacant axial sites displayed cytotoxic activity and nuclear accumulation whereas complexes in which the axial positions were blocked showed little to no toxicity nor uptake.
- ItemOpen AccessRole of Prolactin in a Preclinical Model of Breast Cancer Mediated Osteolysis.(2019-08-29) Gopinathan, Sesha Gopal; Shemanko, Carrie S.; Moorhead, Greg B. G.; Hollenberg, Morley DonaldAt the advanced stage, breast cancer metastasizes to the bone and initiates the vicious cycle of cancer by actively inducing osteoclast differentiation which causes excessive bone degradation. Prolactin (PRL) is a hormone involved in key functions such as mammary gland development and bone homeostasis. We demonstrated that PRL also stimulates breast cancer mediated osteoclastogenesis. The overall goal of my project is to study the role of PRL in breast cancer mediated bone degradation using mouse models. The breast cancer cells chosen for this study were engineered to emit bioluminescence and secrete PRL (MCF7-BGL hPRL) or not (MCF7-BGL-EV). During cell line characterization, MCF7-BGL-hPRL cells demonstrated better osteoclast differentiation via TRAP+ staining, indicating osteolysis potential. Using western blots, the level of PRL secreted by MCF7-BGL-hPRL and the presence of long isoform of PRLR on both the cell lines were quantified. Further, a slight fold difference in the bioluminescence signal intensity between these two cell line was identified and taken into consideration for in vivo experiments. Simultaneously, the role of PRL in osteoblast differentiation and its facilitation of osteoclast differentiation was studied and it showed no influence. Mouse tibia injected with MCF7-BGL-hPRL and MCF7-BGL-EV cells respectively achieved bioluminescence endpoint at 3 weeks and the microCT analysis at 6 weeks revealed higher bone damage in MCF7-BGL-hPRL injected tibia compared to the MCF7-BGL-EV injected tibia. However, a second experiment where the BLI endpoint reached only at 6 weeks showed relatively less bone loss for the same time point. The cumulative results from both the experiments show significant loss in bone mineral density and difference in trabecular thickness in MCF7-BGL-hPRL injected and its uninjected control tibiae, but no difference was observed between the two cell lines. This in vivo study of PRL induced breast cancer mediated osteolysis was never performed before and the valuable information learned from my study outcome sets a platform for future studies.
- ItemOpen AccessThe effect of sodium thiosulfate on cytotoxicity of a diimine Re(I) tricarbonyl complex(The Royal Society of Chemistry, 2021-04-16) Capper, Miles S.; Enriquez Garcia, Alejandra; Lai, Barry; Wang, Baiwen O.; Gelfand, Benjamin S.; Shemanko, Carrie S.; Jalilehvand, FaridehRecently, diimine Re(I) tricarbonyl complexes have attracted great interest due to their promising cytotoxic effects. Here, we compare the cytotoxicity and cellular uptake of two Re(I) compounds fac-[(Re(CO)3(bpy)(H2O)](CF3SO3) (1) and Na(fac-[(Re(CO)3(bpy)(S2O3)])·H2O (bpy = 2,2?-bipyridine) (2). The Re-thiosulfate complex in 2 was characterized in two solvated crystal structures {Na(fac-[Re(CO)3(bpy)(S2O3)])·1.75H2O·C2H5OH}4 (2 + 0.75H2O + C2H5OH)4 and (fac-[Re(CO)3(bpy)(H2O)]) (fac-[Re(CO)3(bpy)(S2O3)])·4H2O (3). The cytotoxicity of 1 and 2 was tested in the MDA-MB-231 breast cancer cell line and compared with that of cisplatin. The cellular localization of the Re(I) complexes was investigated using synchrotron-based X-ray fluorescence microscopy (XFM). The results show that replacement of the aqua ligand with thiosulfate renders the complex less toxic most likely by distrupting its cellular entry. Therefore, thiosulfate could potentially have a similar chemoprotective effect against diimine fac-Re(CO)3 complexes as it has against cisplatin.
- ItemOpen AccessThe Role of Prolactin in the Cellular Response to DNA Damaging Agents(2015-09-30) Karayazi Atici, Odul; Shemanko, Carrie S.High serum levels of the peptide hormone prolactin are associated with increased breast cancer risk and poor prognosis. Prolactin is also involved in breast cancer resistance to different chemotherapeutics. The overall goal of this project is to investigate potential pathways involved in prolactin induced resistance to DNA damaging agents with the hypothesis that the cross-talk between the prolactin pathway and the DNA damage response is important in the mechanism. We previously identified that one isoform of heat shock protein-90 (HSP90), Hsp90alpha, is a prolactin-Janus kinase-2 (Jak2)-signal transducer and activator of transcription-5 (Stat5) regulated gene in breast cancer cells. We have now observed that prolactin increased the viability of breast cancer cells to DNA damaging chemotherapeutics, and Hsp90 inhibitors, 17AAG and BIIB021, abrogated the effect of prolactin, indicating the mechanism of enhanced viability involves the master cancer chaperone Hsp90. The stability of Jak2 and both the total ataxia-telangiectasia mutated protein (ATM) and phospho-ATM appear to be dependent on functional Hsp90. Inhibition of Jak2 and ATM, with highly selective inhibitors (G6 and KU55933, respectively), abrogated prolactin enhanced viability, suggesting their role in prolactin induced cell viability. Drug combination experiments with Hsp90 inhibitor BIIB021 and doxorubicin, and ATM inhibitor KU55933 and doxorubicin, showed drug synergism between doxorubicin and both KU55933 and BIIB021 in MCF7 breast cancer cells. Interestingly, in orthotopic xenograft studies, autocrine prolactin from human breast cancer cells increased the tumor latency of doxorubicin induced DNA damaged cells in SCID mice compared to untreated or prolactin or doxorubicin alone. We hypothesize that this is in part due to the cross-talk of the prolactin and DNA damage response pathways that may be affecting the tumor microenvironment.
- ItemOpen AccessTranscription factors, cofactors and target genes mediating prolactin signals(Kluwer Academic Publishers, 2001) Shemanko, Carrie S.; Groner, Bernd